Effective treatment of many infectious diseases is limited by ignorance of the infected environment and how pathogens adapt to it and alter it. Unfortunately, methods to conduct in situ studies that would provide such insight are largely absent from biomedicine, and there is an urgent need to develop new technical approaches. The existing, dominant paradigm in infectious disease research is to use (necessarily imperfect) reductionist experiments with model organisms to study their physiology and thereby identify new drug targets. Yet ecosystems are much more than the sum of their parts, and interactions (both competitive and cooperative) between species can significantly affect the behavior of individuals. Because of this, traditional approaches likely reveal only part of the bigger picture. Growing awareness of the importance of the human microbiome in determining human health and disease has resulted in an increased appreciation for microbial ecology, yet studies in this area still have focused almost entirely on surveying the microbial communities present within various parts of the body (e.g. the NIH-funded Human Microbiome Project). Here we proposed to develop and apply a number of new approaches to answer the following questions about opportunistic pathogens in the lungs of cystic fibrosis (CF) patients: i) who is there, and how are they spatially associated with each other and with the host? ii) how active are they, as a function of both space and time? and iii) what metabolic pathways are they utilizing? In doing so we will draw on our experience in the field of geobiology, using tools originally developed to track and understand microbes in remote habitats like deep-sea sediments. We will employ state- of-the-art microelectrodes to characterize the chemical environment of the host lung at micrometer length scales, looking for chemical gradients that shape the behavior of the microbial ecosystem and/or are generated by it. Incorporation of 2H from water into lipids will be developed as a novel proxy for in situ growth rates, and spatial mapping of 15N incorporation into proteins using nanoSIMS will be used to discern sub-micrometer patterns of metabolic activity. Fluorescence in situ hybridization (FISH) using 16S rRNA-directed probes will enable the spatial organization between different bacterial species within biofilms to be determined at the level of single cells. Finally, we will develop new FISH probes targeting particular mRNA transcripts using a novel hybridization chain-reaction technique to map metabolic gene expression. These methods will be refined and validated using planktonic and biofilm cultures of Pseudomonas aeruginosa and subsequently applied to CF patient samples in collaboration with clinicians from Children's Hospital L.A. and the USC Adult CF Clinic. Expectorated sputum will be used to gain insight into the temporal evolution of the lung microbiome, whereas explanted lungs will be used to study its spatiometabolic organization. While the CF microbiome will serve as our starting point for the development of these new methods, they ultimately have the potential to transform the study of diverse types of infectious diseases ranging from tuberculosis to malaria.